Pharmacological Treatments for Congenital Myasthenic Syndromes Caused by COLQ Mutations

Background Congenital myasthenic syndromes (CMS) refer to a series of inherited disorders caused by defects in various proteins. Mutation in the collagen-like tail subunit of asymmetric acetylcholinesterase (COLQ) is the second-most common cause of CMS. However, data on pharmacological treatments are limited. Objective In this study, we reviewed related reports to determine the most appropriate pharmacological strategy for CMS caused by COLQ mutations. A literature review and meta-analysis were also performed. PubMed, MEDLINE, Web of Science, and Cochrane Library databases were searched to identify studies published in English before July 22, 2022. Results A total of 42 studies including 164 patients with CMS due to 72 different COLQ mutations were selected for evaluation. Most studies were case reports, and none were randomized clinical trials. Our meta-analysis revealed evidence that β-adrenergic agonists, including salbutamol and ephedrine, can be used as first-line pharmacological treatments for CMS patients with COLQ mutations, as 98.7% of patients (74/75) treated with β-adrenergic agonists showed positive effects. In addition, AChEIs should be avoided in CMS patients with COLQ mutations, as 90.5% (105/116) of patients treated with AChEIs showed either no or negative effects. Conclusion (1) β-adrenergic agonist therapy is the first pharmacological strategy for treating CMS with COLQ mutations. (2) AChEIs should be avoided in patients with CMS with COLQ mutations.

Was follow-up long enough for outcomes to occur?Reporting: 8. Is the case(s) described with sufficient details to allow other investigators to replicate the research or to allow practitioners make inferences related to their own practice?

Eligibility criteria
5 Case reports and studies were included if they fulfilled the following criteria: (1) English-language articles; (2) patients with COLQ mutations with no restriction regarding their age, sex, ethnicity, and treatment; and (3) genetic tests confirmed the COLQ mutation regardless of the details of the mutations.The exclusion criteria were as follows: (1) articles not reporting pharmacological treatment(s) and ( 2) articles that did not mention the treatment effect of a pharmacological therapy.

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Information sources 6 The literature search was restricted to articles published in English.We searched the PubMed (1966-2022), MEDLINE (1950-2022), Web of Science (1864-2022), and Cochrane Library (2022) databases.The most recent search was performed on July 22, 2022.In addition, a manual search was performed to identify references in the obtained studies to identify other possible studies.

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Search strategy 7 The keywords "COLQ mutations," "COLQ," or "congenital myasthenic syndrome, COLQ" were used.Page 3 Selection process 8 The studies were read thoroughly to assess their eligibility for inclusion in the meta-analysis.SS and GS extracted data and performed the analysis.The quality of individual studies was assessed using the recently published "Tool for evaluating the methodological quality of case reports and case series" proposed by Murad et al. [36] Based on the previous criteria of the Pierson, Bradford Hills, and Newcastle-Ottawa scale.Each study was independently evalua-

# Checklist item
Location where item is reported ted according to four domains (selection, ascertainment, causality, and reporting) to yield an overall assessment (Supplementary Table S2).The quality assessment included eight leading exploratory questions with a binary response (yes/no) to determine whether the items suggested the presence of bias.
Effect measures 12 Data analysis was performed using Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) version 25.Statistical differences were estimated using the Kruskal-Wallis test or one-way analysis of variance followed by the Bonferroni post-hoc test.The analysis of the relationship between sex and treatment effect was estimated using the Mann-Whitney test (two groups).Simple linear regression was used to analyze the relationship between age of onset and treatment effect.Statistical significance was set at p < 0.05.

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Synthesis methods 13a The following data were extracted directly from the articles: clinical information, gene mutations, pharmacological treatments, and follow-up data.Clinical data including sex, age at symptom onset, and age at the time of the case study were reported.If more than one patient had been reported in different studies or by the same group, the participants in the studies were compared.Data that appeared more than once were excluded from the analysis.

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The quality of individual studies was assessed using the recently published "Tool for evaluating the methodological quality of case reports and case series" proposed by Murad et al. [36] Based on the previous criteria of the Pierson, Bradford Hills, and Newcastle-Ottawa scale.Each study was independently evaluated according to four domains (selection, ascertainment, causality, and reporting) to yield an overall assessment (Supplementary Table S2).The quality assessment included eight leading exploratory questions with a binary response (yes/no) to determine whether the items suggested the presence of bias.
Page 3 13c Statistical differences were estimated using the Kruskal-Wallis test or one-way analysis of variance followed by the Bonferroni post-hoc test.The analysis of the relationship between sex and treatment effect was estimated using the Mann-Whitney test (two groups).Simple linear regression was used to analyze the relationship between age of onset and treatment effect.Statistical significance was set at p < 0.05.
Page 3 13d Data analysis was performed using Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) version Page 3 13e Statistical differences were estimated using the Kruskal-Wallis test or one-way analysis of variance followed by the Bonferroni post-hoc test.The analysis of the relationship between sex and treatment effect was estimated using the Mann-Whitney test (two groups).Simple linear regression was used to analyze the relationship between age of onset and treatment effect.Statistical significance was set at p < 0.05.
Page 3 13f Statistical differences were estimated using the Kruskal-Wallis test or one-way analysis of variance followed by the Bonferroni post-hoc test.The analysis of the relationship between sex and treatment effect was estimated using the Mann-Whitney test (two groups).Simple linear regression was used to analyze the relationship between age of onset and treatment effect.Statistical significance was set at p < 0.05.

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Reporting bias assessment 14 Each study was independently evaluated according to four domains (selection, ascertainment, causality, and reporting) to yield an overall assessment (Supplementary Table S2).

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Certainty assessment 15 The studies were read thoroughly to assess their eligibility for inclusion in the meta-analysis.SS and GS extracted data and performed the analysis.

Study selection 16a
After searching, 42 studies meeting our inclusion and exclusion criteria were selected, including 164 patients with CMS due to 72 different COLQ mutations [6, 12-14, 17, 19, 22, 27-30, 37-61].Most selected studies were case reports, and none were RCTs.The information for each patient is listed in Supplementary Table S3, including geographical settings, mutation, onset age, related risk factors, treatment, and effects.
Page 4 16b All the studies matching our including criteria and not matching our excluding criteria were included.Page 4 Study characteristics 17 Each study was independently evaluated according to four domains (selection, ascertainment, causality, and reporting) to yield an overall assessment (Supplementary Table S2).

# Checklist item
Location where item is reported evaluation.Most studies were case reports, and none were randomized clinical trials.Our meta-analysis revealed evidence that β-adrenergic agonists, including salbutamol and ephedrine, can be used as first-line pharmacological treatments for CMS patients with COLQ mutations, as 98.7% of patients (74/75) treated with β-adrenergic agonists showed positive effects.In addition, AChEIs should be avoided in CMS patients with COLQ mutations, as 90.5% (105/116) of patients treated with AChEIs showed either no or negative effects.
20c However, there were some limitations to our meta-analysis.(1) There might have been publication bias due to the possibility that only positive results were published.( 2) Most reports that we reviewed lacked aquantitative assessment of treatment effectiveness.Instead, they were described qualitatively using words such as negative, partial, and positive.This may also lead to bias and flatten variations in reported treatment outcomes.(3) Insufficient information was provided regarding drug doses, so it was difficult to discern dose-effect relationships for the investigated treatments.
Page 4,5 20d Forest plot was not made due to the special method used to analysis.Page 5 Reporting biases 21 However, there were some limitations to our meta-analysis.(1) There might have been publication bias due to the possibility that only positive results were published.( 2) Most reports that we reviewed lacked aquantitative assessment of treatment effectiveness.Instead, they were described qualitatively using words such as negative, partial, and positive.This may also lead to bias and flatten variations in reported treatment outcomes.(3) Insufficient information was provided regarding drug doses, so it was difficult to discern dose-effect relationships for the investigated treatments.
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Page 5 23b However, there were some limitations to our meta-analysis.(1) There might have been publication bias due to the possibility that only positive results were published.(2) Most reports that we reviewed lacked aquantitative assessment of treatment effectiveness.Instead, they were described qualitatively using words such as negative, partial, and positive.This may also lead to bias and flatten variations in reported treatment outcomes.(3) Insufficient information was provided regarding drug doses, so it was difficult to discern dose-effect relationships for the investigated treatments.

Discussion
Page 8 23c However, there were some limitations to our meta-analysis.(1) There might have been publication bias due to the possibility that only positive results were published.(2) Most reports that we reviewed lacked aquantitative assessment of treatment effectiveness.Instead, they were described qualitatively using words such as negative, partial, and positive.This may also lead to bias and flatten variations in reported treatment outcomes.(3) Insufficient information was provided regarding drug doses, so it was difficult to discern dose-effect relationships for the investigated treatments.
Page 8 23d However, there were some limitations to our meta-analysis.(1) There might have been publication bias due to the possibility that only positive results were published.(2) Most reports that we reviewed lacked aquantitative assessment of treatment effectiveness.Instead, they were described qualitatively using words such as negative, partial, and positive.This may also lead to bias and flatten variations in reported treatment outcomes.(3) Insufficient information was provided regarding drug doses, so it was difficult to discern dose-effect relationships for the investigated treatments.For more information, visit: http://www.prisma-statement.org/

Supplementary Figure 1 . 3 Objectives 4
IM/NPD/I/MD/V refers to intermarriage/normal pregnancy and delivery/experience of respiratory infections/motor function developmental delay/assisted ventilation.Y and N means yes and no.0 means the information isn't mentioned.In the line of treatment, the drug type and effect are listed.The drugs include acetylcholinesterase inhibitors (AChEIs), β-adrenergic receptor agonists (BAs), 3,4-diaminopyridine (DAP), fluoxetine (FLX), glucocorticosteroids (GCs) and other drugs.Other drug A (OA) refers to L-Carnitine and Coenzyme Q10.Other drug B (OB) refers to cocktail of carnitine, thiamine, riboflavin, menadione and Coenzyme Q.Other drug C (OC) refers to the combination of levetiracetam, carbamazepine, midazolam, clonazepam, vitamin B6, and vigabatrin.Treatment effect is categorized into four types: (-) no effect, negative or ambiguous response; (+) partial, incomplete, moderate, modest/mild effect or denotes initial positive response followed by ineffectiveness; (++) beneficial, positive or clear effect; (+++) remarkable, dramatical or satisfying effect.Influence of Mutation Type on the Pharmacological Treatment Effects.The treatment effect was categorized into four types: (-) no effect, negative or ambiguous response; (+) partial, incomplete, moderate, modest/mild effect or denotes initial positive response followed by ineffectiveness and worsening; (++) beneficial, positive or clear effect; (+++) remarkable, dramatical or satisfying effect.Patients are divided in to different groups according to the COLQ mutation types: missense/missense, missense/truncating, truncating/truncating.Each point represents a patient.Mean and SEM are indicated.No significant difference was found by Kruskal-Wallis test (p > 0.05).INTRODUCTION Rationale 3 Different therapies have been applied for treating CMS in clinical settings.However, due to the interactions of various mechanisms, the same drug may have varying or even opposite effects on different CMS subtypes, which makes it necessary to analyze pharmacological treatments based on disease subtypes.Here, we reviewed related reports and attempted to determine the most appropriate pharmacological strategy for CMS caused by COLQ mutations.Page To determine the most appropriate pharmacological strategy for CMS caused by COLQ mutations.Page 3

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review has not been registered in the international prospective register of systematic reviews (PROSPERO).Page This systematic review and meta-analysis protocol has not been published.Page This systematic review has not been registered in the international prospective register of systematic reviews (PROSPERO).Page This work was supported by the Science and Technology Innovation Program of Hunan Province, China (Grant No. 2021RC2023, KH), the China Postdoctoral Science Foundation (Grant No. 2021M703638, KH), and the National Natural Science Foundation of China (Grant No. 82201557, KH).

Table 3 ,
we listed the information of each patient.GS means geographical settings of each patient.HIR refers to high-income-regions; LMR refers to low-and middle-income regions.In the description of mutation position, E refers to exon and I refers to intron.Onset Age refers to the age when symptoms of CMS appear.EI refers to early infancy.NA refers to not available information.
23a In our study, β-adrenergic receptor agonists performed best among all pharmacological methods for treating patients with CMS due to COLQ mutations.These drugs are widely used in clinical practice.The beneficial effect of ephedrine inpatients with myasthenia gravis has been recognized since the 1930s[70].Physicians reported its effect on improvingmuscle strength and decreasing fatigability when treating dysmenorrhea by chance [71, 72], but anticholinesterases and corticosteroids have since replaced it owing to possible adverse effects[73].Ephedrine returned to the spotlight again owing to its effect on CMS treatment, especially CMS caused by DOK7 mutations.DOK7 is a muscle-intrinsic activator of MuSK.
The information for each patient is listed in Supplementary TableS3, including geographical settings, mutation, onset age, related risk factors, treatment, and effects.From: Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al.The PRISMA 2020 statement: an updated guideline for reporting systematic reviews.BMJ 2021;372:n71.doi: 10.1136/bmj.n71